Extensive multiplexing of protein nucleic-acid interactions to comprehensively study gene expression regulation from chromatin to mRNA degradation

项目摘要 基因表达在RNA和蛋白质水平上都受到染色质机制的严格控制 修饰、转录调节、mRNA剪接、加工、翻译和降解。这一切成功都 这些过程受核酸-蛋白质相互作用（DNA-蛋白质和RNA-蛋白质）的调节。因此， 科学界一直在努力全面绘制这些相互作用，包括 主要的国际研究工作（如ENCODE、RoadMap Epigenomics）侧重于生成参考 特定细胞类型的映射。然而，由于这些结合图谱对单个细胞类型具有高度特异性， 迫切需要为任何感兴趣的细胞类型生成全面的基因组图谱- 包括原代细胞类型、疾病模型或其他稀有细胞群。这一目标 仍然具有挑战性，因为现有的测定法一次只能绘制单一蛋白质的相互作用， 因此非常昂贵。 为了解决这些问题，该提案将开发一种高度创新的技术， 使用高通量绘制多途径蛋白质-核酸相互作用的条形码化策略（SPRITE） 测序所提出的Hi-P技术将用于建立：（i）高度多重的eCLIP-seq方法 将多达数百种RNA结合蛋白同时映射到它们的RNA结合位点，（ii）高度多重的 ChIP-seq方法可映射多达数百种DNA结合蛋白及其DNA结合的组蛋白修饰 位点，以及（iii）在许多样品中绘制这些多种蛋白质-核酸相互作用的方法， 这些罕见的细胞类型 拟议中的技术代表了一个重大进步-它将大大增加现有方法的规模 并创造目前不可能的新能力。这些工具将使个人研究人员能够 在特定的生物和疾病背景下生成详细的基因组数据集，这些数据集在大小上是可比的， 与ENCODE项目所产生的复杂性相比，其成本只有ENCODE项目的一小部分。更一般地说，我们预计 这些工具将为基因调控和人类疾病带来重要的新见解。

Project Summary Gene expression is tightly controlled at both the RNA and protein level by mechanisms involving chromatin modification, transcriptional regulation, mRNA splicing, processing, translation and degradation. Each of these processes are regulated by nucleic acid-protein interactions (DNA-protein and RNA-protein). Accordingly, there have been tremendous efforts in the scientific community to comprehensively map these interactions, including major international research efforts (e.g. ENCODE, RoadMap Epigenomics) focused on generating reference maps for specific cell types. However, because these binding maps are highly specific for individual cell types, there is a critical need to enable the generation of comprehensive genomic maps for any cell type of interest – including primary cell types, disease models, or other rare cell populations – within an individual lab. This goal remains challenging because existing assays can only map interactions of a single protein at a time and are therefore prohibitively expensive. To address these issues, this proposal will develop a highly innovative technology based on our split-pool barcoding strategy (SPRITE) that maps multiway protein-nucleic acid interactions using high throughput sequencing. The proposed Hi-P technology will be used to establish: (i) a highly multiplexed eCLIP-seq method to map up to hundreds of RNA binding proteins simultaneously to their RNA binding sites, (ii) a highly multiplexed ChIP-seq method to map up to hundreds of DNA binding proteins and histone modifications to their DNA binding sites, and (iii) methods to map these multiple protein-nucleic acid interactions across many samples, among these rare cell types, simultaneously. The proposed technology represents a major advance – it will dramatically increase the scale of existing methods and create new capabilities that are currently not possible. These tools will empower individual researchers to generate detailed genomic datasets in specific biological and disease contexts that are comparable in size and complexity to those generated by the ENCODE project at a tiny fraction of its cost. More generally, we anticipate that these tools will lead to critical new insights into gene regulation and human disease.